The present invention relates generally to the field of proteins associated with neural lesions More specifically, the present invention relates to the purification, detection and methods of use of protease nexin-2 (hereinafter "PN-2").
This invention was made with Government support under Grant No. GM-31609 awarded by the National Institutes of Health. The Government has certain rights in this invention. American Cancer Society Grants CD 390 and BC 602/BE 22A provided further support for the development of this invention.
The protease nexins are protein proteinase inhibitors that are synthesized and secreted by a variety of cultured extravascular cells. PN-2 is a soluble, acidic protein fragment which forms stoichiometric, inhibitory complexes with several serine proteinases including, but not limited to, epidermal growth factor binding protein (EGF BP), the gamma subunit of 7S nerve growth factor (NGF-.gamma.) and trypsin. In contrast to most serine proteinase inhibitors, PN-2 is very stable, retaining its inhibitory activity after treatments with SDS and pH 1.5.
PN-2 is believed to be derived from the .beta.-amyloid precursor protein (hereinafter ".beta.APP"). .beta.APP is known to be associated with neural lesions of Alzheimer's disease and Down's syndrome. .DELTA.APP comprises about 695 amino acid residues and has the characteristics of a cell receptor protein, comprising cytoplasmic, transmembrane and extracellular domains. Alternatively spliced forms of this precursor protein further comprise a 57 residue insert which is homologous to that of a "Kunitz-type" protease inhibitor which inhibits trypsin.
We have previously purified PN-2 from serum-free culture medium that was conditioned by human fibroblasts. However, this material was slightly contaminated with other components. Thus, there is a need for an improved method for purifying PN-2.
Alzheimer's disease is characterized by the accumulation of amyloid protein both intracellularly and extracellularly in the tissues of the brain, notably in neuritic plaques. The major protein subunit of these amyloid plaques has been identified as a polypeptide of about 4.5 kD having the ability to aggregate. This protein subunit is variously referred to as the amyloid .beta.-protein or as amyloid A4, and is herein referred to as "A4".
A4 is thought to have its origin, through proteolytic cleavage, in .DELTA.APP. Release of the A4 unit is thought to occur by proteolysis of the precursor which may result from membrane damage. Because A4 is believed to be critical to the formation of amyloid plaques, there is a need for methods of preventing the release of A4.
The cDNA for .DELTA.APP lacking the Kunitz domain has been cloned and the nucleotide sequence determined. From the nucleotide sequence, the amino acid sequence has been predicted. The A4 peptide lies at residues 597 to 648 of the deduced amino acid sequence.
Alzheimer's disease produces a debilitating dementia for which no treatment is known. Thus, there is a need for methods of treatment and prevention of this disease.
Down's syndrome is a genetic disease which usually causes mental retardation and other symptoms. An unusually high number of people with Down's also develop Alzheimer's after the age of 40. Thus, there is a need for a treatment for Down's.
Presently, definitive diagnosis of Alzheimer's disease is only available at autopsy. Such diagnosis involves examination of brain tissue for extracellular neuritic plaques and intracellular tangles of microtubule-associated proteins and other cytoskeletal elements. The plaques are believed to start to form years before any clinical sign of Alzheimer's appears.
Many researchers believe that there is a correlation between the density of neuritic plaques and the severity of dementia. Thus, there is a need for preventing further development of neuritic plaques. If applied prior to the development of Alzheimer's symptoms, such a treatment, may thereby prevent onset of the disease.
Even without a treatment for Alzheimer's, an early diagnosis of the disease would allow physicians to rule out other causes of dementia. Moreover, in order to study the genetics of Alzheimer's it would be very useful to obtain a diagnosis of the disease prior to autopsy. More importantly, once treatments are developed, an early diagnosis may prove critical in the treatment's ability to improve mental functioning. Thus, there is a need for a method for the early diagnosis of Alzheimer's.